There are generally recognised to be three types of organ rejection: hyperacute, acute and chronic. Hyperacute rejection generally occurs within twenty-four hours of the transplantation, and is readily detected. Acute rejection is generally regarding as rejection occurring within the first six months of transplantation. Acute rejection can be diagnosed relatively easily, for example, in the case of a cardiac transplant by the appearance of certain cell types in biopsy cell infiltrate, and in the case of kidney and liver transplants by the change in the levels of certain serum enzymes. Chronic rejection, generally regarded as that occurring at least six months after transplantation, is very difficult to diagnose clinically, and may not manifest itself clearly for some years, by which time treatment is generally unsuccessful.
In chronic rejection there is typically vasculopathy in the rejected organ. Transplant associated coronary artery disease (TxCAD), a rapidly progressing obliterative vascular disease developing in transplanted heart, is the most important complication after the first year of cardiac transplantation, with an incidence of 40% at five years post transplant. A similar vasculopathy occurs following kidney transplantation where it is designated chronic rejection. Higher incidences of the disease are reported using intravascular ultrasound. Histologically, the grafted vessels become occluded with an intimal lesion consisting of smooth muscle cells, myofibroblasts and deposition of extracellular matrix proteins.
The aetiology of TxCAD remains ill defined and several immunological and non-immunological factors have been reported to associate with the risk of TxCAD. This complicated aetiology means that TxCAD remains difficult to diagnose clinically. The denervated heart, for example, prevents anginal symptoms and the diffuse concentric distribution of the lesions can obscure angiographic evidence of stenosis. Chronic rejection of other organs can also be very difficult to diagnose clinically. For example, in the case of renal transplants, rejection cannot be distinguished from cyclosporin nephrotoxicity.
Immunological damage to transplanted organs continues to be the major complication and significant cause of morbidity and mortality, especially following cardiac transplantation. T cells have been described immunocytochemically beneath the endothelium in atherosclerotic plaques from patients with accelerated coronary artery disease. It is likely, however, that T cells invade the endothelium at an early stage of the disease long before there is angiographic evidence of abnormalities. Integrity of the endothelium is recognised as being a crucial factor in maintaining normal vessel function and endothelial injury is probably the earliest event which initiates all forms or arteriosclerosis. Anti-endothelial antibodies can be highly destructive, for example they cause rapid rejection of xenografted organs. Although there is compelling evidence from experimental models to support a pathogenic role of antibodies in chronic rejections, the association in humans remains far from clear, in particular regarding the specificity of antibodies made after transplantation and whether in fact such antibodies can damage graft tissues.
Until recently, endothelial autoantigens have been characterised using Western blotting. Western blotting methods separate endothelial peptides by one dimensional gel electrophoresis. This method can only display a limited number of antigens because many different peptides, having the same molecular weight but different electrophoretic charges, will appear in the same band. A better approach is to separate endothelial peptides by charge and molecular weight using 2-D electrophoresis and probe the subsequent blots with patient sera.
We have previously used these techniques to identify approximately 40 immunoreative cytosolic proteins of which around 30% could be identified by end terminal amino acid sequencing. The most abundant immunoreactive antigen was identified as being the intermediate filament vimentin, see U.S. Pat. No. 5,716,787 and Wheeler et al, 1995. Accordingly, these references disclose that the cytoskeletal protein vimentin or anti-vimentin antibodies are markers that can be used in the diagnosis of TxCAD or chronic rejection in renal transplantation.
However, 2-D electrophoresis suffers from the problem that it can lack the sensitivity to detect the most immunoreactive proteins as they may not be abundant enough to allow their chemical characterisation. Accordingly, it remains a continuing problem in the art to find markers which can provide an accurate and early diagnosis of chronic rejection.